鸡白细胞介素18单克隆抗体的制备与鉴定  

Preparation and Identification of Monoclonal Antibodies against chIL-18

作　　者：郑孟加 马萌 王永强[1] 高丽 曹红[1] 李淑芹[1] 李晓齐[1] 郑世军[1] ZHENG Meng-jia;MA Meng;WANG Yong-qiang;GAO Li;CAO Hong;LI Shu-qin;LI Xiao-qi;ZHENG Shi-jun(Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs,College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]中国农业大学动物医学院农业农村部动物流行病学重点实验室,北京海淀100193

出　　处：《中国兽医杂志》2022年第9期29-33,共5页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(32130105);现代农业产业技术体系建设专项(CARS-40)。

摘　　要：为制备鸡白细胞介素18(chIL-18)单克隆抗体,本试验从被沙门菌感染过的鸡脾脏DNA提取物中扩增chIL-18基因,并将其插入至原核表达载体pET-28a和pGEX-6p-1中,在大肠埃希菌中诱导表达并纯化重组蛋白;继而将纯化后的His-chIL-18蛋白免疫BALB/c小鼠,经3次免疫后,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合,以GST-chIL-18作为筛选蛋白通过间接ELISA方法对阳性杂交瘤细胞进行筛选,经3次亚克隆后,最后获得2株能稳定分泌IL-18抗体的杂交瘤细胞,分别命名为2A7和2G5。对抗体纯化后进行鉴定,结果显示,2株抗体亚型均为IgG1,亲和力解离常数(Kd)分别为4.4×10^(-12)和2.06×10^(-10),与chIL-1、chIL-10和chIFN-β无交叉反应,可分别识别chIL-18第58~113位和第114~169位氨基酸区域。本试验制备的单抗为深入研究chIL-18的生物学功能及先天性免疫应答提供了有价值的手段。To prepare monoclonal antibody(McAb)against chicken interleukin-18(chIL-18),the chIL-18 gene was amplified from chicken splenic cells DNA infected with Salmonella and inserted into prokaryotic expression vectors pET-28a and pGEX-6p-1,the recombinant proteins were induced and purified in Escherichia coli.Then,the purified recombinant His-chIL-18 protein was used as an immunogen to immunize BALB/c mice.After three immunizations,murine splenic cells were fused with myeloma cells(SP2/0)in vitro,and GST-chIL-18 was used as a coating protein in indirect ELISA to screen positive hybridoma cells secreting anti-chIL-18 antibodies.Two hybridoma cells capable of stably secreting IL-18 antibodies were obtained after three rounds of subcloning,and named 2A7 and 2G5,respectively.Both monoclonal antibodies were identified as IgG1 subtype,with the affinity constants of dissociation(Kd)of 4.4×10^(-12) and 2.06×10^(-10),respectively,and displayed no cross-reaction with chIL-1、chIL-10 and chIFN-β.The epitope domains of chIL-18 recognized by these two monoclonal antibodies were determined to be at positions 58-113 aa and 114-169 aa,respectively.The development of two monoclonal antibodies against chIL-18 offers a valuable approach for further study of the biological function and innate immune response to chIL-18.

关 键 词：鸡IL-18 原核表达 单克隆抗体 

分 类 号：S852.4[农业科学—基础兽医学]